The goal of this research is to define the mechanism(s) by which the amoeba, Naegleria fowleri, develops resistance to macrophage cytolytic molecules. In conducting these studies, N. fowleri LEE (a weakly pathogenic strain) and N. fowleri LEEmp ( a highly pathogenic mouse- passaged strain) will be employed. Transition to the highly pathogenic state occurs when amoebae are passaged through mice. This transition is accompanied by increased resistance to lysis by macrophage cytolytic factors (M0CF's). Recombinant-corynebacterium parvum-activated M0's. The interaction of M0CF's with the amoebae will be examined at the ultrastructural level to determine whether damage to the amoeba membrane or internal organelles occurs. The second specific objective is to purify and characterize membrane components and metabolic products of N. fowleri will be utilized to compare changes in protein profiles using two- patterns of the two strains will be detected by surface iodination of intact organisms. Lectin precipitation of 125 I-surface labeled proteins followed by SDS-PAGE and staining with Periodic Acid Schiff reagent or sudan black will permit identification of surface glycoproteins or glycolipids. Cell fractionation of amoebae will be performed using Percoll gradients. Resultant membrane fractions will be further purified by high performance liquid chromatography. Fractions eluted from HPLC columns will be analyzed by SDS-PAGE. Metabolic products of both strains will be similarly analyzed. The third specific objective is to determine whether purified membrane components are resistant to degradation by M0GF's or TNF. Membrane components will be treated with M0CF's or TNF and the amoebae. The fourth specific objective is to determine whether amoebae membrane components, such as proteases, or metabolic products inactivate M0CF's or TNF. The fifth specific objective is to determine whether specific membrane components of highly pathogenic amoebae are shed in response to treatment with M0CF's or TNF. 125 O-surface components in medium containing amoebae treated with M0CF's will be measured.